Background and objectives: Toxigenic Vibrio cholerae contains ctxA, ctxB and zot genes and comparing to non-toxigenic strains causes severe disease in human. Therefore accurate identification of toxigenic strains is very important. This study the usage of a Multiplex PCR assay for simultaneous detection of the ctxA,ctxB and zot genes of V. cholerae isolated from patients was investigated.Material and Methods: In this study, 73 stool samples were collected and were determined as V. choleraeO1 by serological and biochemical tests. Finally, all of strains. Finally the results were analyzed statistically by SPSS 16 software. Results: According to the results of the Multiplex PCR, the incidences of ctxA , ctxB and zot genes in clinical isolates were obtained as 90.4% (66 samples), 90.4% (66 samples), and 91.7% (67 samples), respectively. All of the strains were belonged to O1serogroup. The results showed that Multiplex PCR is a method for the rapid and accurate detection for toxigenic V. cholerae in clinical isolates. Also, this assay is specific for the detection of Vibrio strains that possess the ctxA, ctxB and zot genes with sensitivity up to 1/100 dilution of the genome per reaction. Conclusion: In case where specific primers were utilized (target genes of ctxA, ctxB and zot ), Multiplex PCR has proved to be a simple, fast, and relatively inexpensive method for the rapid and accurate detection of toxigenic V. cholerae strains in the clinical samples.